If DNA double-strand breaks (DSBs) are repaired improperly, they may lead to chromosome translocations and cancer. DSBs are created by exogenous agents such as ionizing radiation (IR), or by the endogenous process of V(D)J recombination, which rearranges immunoglobulin and T cell receptor genes to generate immunological diversity. Remarkably, mammalian cells utilize the same end-joining reaction for both DSB repair and V(D)J recombination. The end-joining reaction must include steps for synapsis of two DNA ends, processing of the ends for alignment by microhomology base pairing, and ligation into a covalently linked DNA duplex. Proteins required for this process have been identified by this and other laboratories and include DNA-dependent protein kinase (DNA-PK) and XRCC4. DNA-PK consists of a regulatory subunit, Ku, which binds to DNA ends, and a catalytic subunit, p460, which is activated by DNA ends. XRCC4 may act late in the pathway by stimulating the final ligation reaction. A model has been formulated for how Ku and p460 participate in end-joining. To test the model, the specific aims of this proposal are to investigate: 1. The roles of Ku and p460 in the synapsis of DNA ends. A super-large complex of DNA, Ku and p460 was detected and will be characterized to determine if it represents the synapsis of 2 DNA molecules. A 19 Angstrom resolution structure of p460 was obtained, suggesting synapsis may occur in a parallel orientation. Experiments will distinguish between parallel and antiparallel synapsis. 2. The roles of Ku and p460 in the processing of DNA ends. DNA-PK phosphorylation of Ku activates its ATPase activity, which will be tested for possible roles in processing DNA ends: strand exchange, end-unwinding, unwinding coupled to microhomology base pairing, and opening hairpin ends. Ku mutated in its ATP binding motifs will be transfected into Ku mutant cells to test the role of the ATPase in vivo. 3. Later steps in the end-joining reaction. The complex of Ku and p460 on DNA ends was discovered to recruit additional factors. These factors will be identified by protein purification and by testing specific candidates, including XRCC4, DNA ligases I and IV, RPA, HMG1 and HMG2. Once the initial steps have been characterized, a search will be conducted for the complete end-joining reaction in vitro. Ultimately, these studies will lead to a molecular description of both DSB repair and V(D)J recombination, leading to deeper insight into both cancer and the human immune system.